Mutations in the NB-ARC domain of I-2 that impair ATP hydrolysis cause autoactivation

Resistance (R) proteins in plants confer specificity to the innate immune system. Most R proteins have a centrally located NB-ARC (nucleotide-binding adaptor shared by APAF-1, R proteins, and CED-4) domain. For two tomato (Lycopersicon esculentum) R proteins, I-2 and Mi-1, we have previously shown that this domain acts as an ATPase module that can hydrolyze ATP in vitro. To investigate the role of nucleotide binding and hydrolysis for the function of I-2 in planta, specific mutations were introduced in conserved motifs of the NB-ARC domain. Two mutations resulted in autoactivating proteins that induce a pathogen-independent hypersensitive response upon expression in planta. These mutant forms of I-2 were found to be impaired in ATP hydrolysis, but not in ATP binding, suggesting that the ATP- rather than the ADP-bound state of I-2 is the active form that triggers defense signaling. In addition, upon ADP binding, the protein displayed an increased affinity for ADP suggestive of a change of conformation. Based on these data, we propose that the NB-ARC domain of I-2, and likely of related R proteins, functions as a molecular switch whose state (on/off) depends on the nucleotide bound (ATP/ADP).     

植物TIR-NB-LRR类型抗病基因各结构域的研究进展

植物抗病反应是一个多基因调控的复杂过程,在这个过程中R基因发挥了非常重要的作用。根据其氨基酸基序组成以及跨膜结构域的不同,R基因可以分为多种类型,其中NBS-LRR类型是植物基因组中最大的基因家族之一。TIR-NB-LRR类型的抗病基因又是NB-LRR类型中的一大类,也是目前抗病基因研究的热点。该文总结了TIR-NB-LRR类型抗病基因各个结构域的功能和相关的研究进展。相关研究表明,TIR结构域主要通过自身或异源的二聚体化介导抗性信号的转导,但也有部分研究表明,该结构域可能参与病原菌的特异性识别。NBS结构域常被认为具有"分子开关"的功能,它可以通过结合ADP或ATP来调节植物抗病蛋白的构象变化,从而调节下游抗病信号的传导。LRR结构域在植物与病原菌互作的过程中可以通过与病原菌的无毒蛋白直接或间接互作来特异识别病原菌。也有研究发现,LRR结构域具有调节信号传导的功能。这些信息将为研究植物抗病机理提供理论依据,也为将来通过基因编辑技术对作物进行定向抗病育种提供思路。     

An autoactive mutant of the M flax rust resistance protein has a preference for binding ATP, whereas wild-type M protein binds ADP

Resistance (R) proteins are key regulators of the plant innate immune system and are capable of pathogen detection and activation of the hypersensitive cell death immune response. To understand the molecular mechanism of R protein activation, we undertook a phenotypic and biochemical study of the flax nucleotide binding (NB)-ARC leucine-rich repeat protein, M. Using Agrobacterium-mediated transient expression in flax cotyledons, site-directed mutations of key residues within the P-loop, kinase 2, and MHD motifs within the NB-ARC domain of M were shown to affect R protein function. When purified using a yeast expression system and assayed for ATP and ADP, these mutated proteins exhibited marked differences in the quantity and identity of the bound nucleotide. ADP was bound to recombinant wild-type M protein, while the nonfunctional P-loop mutant did not have any nucleotides bound. In contrast, ATP was bound to an autoactive M protein mutated in the highly conserved MHD motif. These data provide direct evidence supporting a model of R protein function in which the "off" R protein binds ADP and activation of R protein defense signaling involves the exchange of ADP for ATP.     

Plant extracellular ATP signalling: new insight from proteomics

Complex signalling systems have evolved in multicellular organisms to enable cell-to-cell communication during growth and development. In plants, cell communication via the extracellular matrix (apoplast) controls many processes vital for plant survival. Secretion of ATP into the extracellular matrix is now recognised as a previously unknown stimulus for cell signalling with a role in many aspects of plant physiology. In the last decade, the secondary messenger molecules in extracellular ATP signalling were identified, but the downstream gene and protein networks that underpin plant responses to extracellular ATP are only beginning to be characterised. Here we review the current status of our knowledge of plant extracellular signalling and demonstrate how applying state-of-the art proteomic technologies is rapidly bringing new discoveries in extracellular ATP research. We discuss how monitoring of the global proteomic profile during responses to modulation of extracellular ATP signalling has led to novel insight into pathogen defence systems and plant programmed cell death regulation. On the basis of extensive proteomic, pharmacological, and reverse genetics data, extracellular ATP has been confirmed to constitute an important molecular switch that tightly controls organellar energy metabolism, reprogramming of primary metabolic pathways, and redirection of resources to protein networks that support adaptation of plants to stress.     

Nucleotide binding to the homodimeric MJ0796 protein: a computational study of a prokaryotic ABC transporter NBD dimer

Transport by ABC proteins requires a cycle of ATP-driven conformational changes of the nucleotide binding domains (NBDs). We compare three molecular dynamics simulations of dimeric MJ0796: with ATP was present at both NBDs; with ATP at one NBD but ADP at the other; and without any bound ATP. In the simulation with ATP present at both NBDs, the dimeric protein interacts with the nucleotides in a symmetrical manner. However, if ADP is present at one binding site then both NBD-NBD and protein-ATP interactions are enhanced at the opposite site.     

Resistance proteins: molecular switches of plant defence

Specificity of the plant innate immune system is often conferred by resistance (R) proteins. Most R proteins contain leucine-rich repeats (LRRs), a central nucleotide-binding site (NBS) and a variable amino-terminal domain. The LRRs are mainly involved in recognition, whereas the amino-terminal domain determines signalling specificity. The NBS forms part of a nucleotide binding (NB)-ARC domain that presumably functions as a molecular switch. The conserved nature of NB-ARC proteins makes it possible to map mutations of R protein residues onto the crystal structures of related NB-ARC proteins, providing hypotheses for the functional roles of these residues. A functional model emerges in which the LRRs control the molecular state of the NB-ARC domain. Pathogen recognition triggers nucleotide-dependent conformational changes that might induce oligomerisation, thereby providing a scaffold for activation of downstream signalling components.     

The effects of extracellular adenosine 5′‐triphosphate on the tobacco proteome

Extracellular adenosine 5′‐triphosphate (eATP) is emerging as an important plant signalling compound capable of mobilising intracellular second messengers such as Ca²⁺, nitric oxide, and reactive oxygen species. However, the downstream molecular targets and the spectrum of physiological processes that eATP regulates are largely unknown. We used exogenous ATP and a non‐hydrolysable analogue as probes to identify the molecular and physiological effects of eATP‐mediated signalling in tobacco. 2‐DE coupled with MS/MS analysis revealed differential protein expression in response to perturbation of eATP signalling. These proteins are in several functional classes that included photosynthesis, mitochondrial ATP synthesis, and defence against oxidative stress, but the biggest response was in the pathogen defence‐related proteins. Consistent with this, impairment of eATP signalling induced resistance against the bacterial pathogen Erwinia carotovora subsp. carotovora. In addition, disease resistance activated by a fungal pathogen elicitor (xylanase from Trichoderma viride) was concomitant with eATP depletion. These results reveal several previously unknown putative molecular targets of eATP signalling, which pinpoint eATP as an important hub at which regulatory signals of some major primary metabolic pathways and defence responses are integrated.     

An ATP/ADP-Dependent Molecular Switch Regulates the Stability of p53-DNA Complexes

Interaction with DNA is essential for the tumor suppressor functions of p53. We now show, for the first time, that the interaction of p53 with DNA can be stabilized by small molecules, such as ADP and dADP. Our results also indicate an ATP/ADP molecular switch mechanism which determines the off-on states for p53-DNA binding. This ATP/ADP molecular switch requires dimer-dimer interaction of the p53 tetramer. Dissociation of p53-DNA complexes by ATP is independent of ATP hydrolysis. Low-level ATPase activity is nonetheless associated with ATP-p53 interaction and may serve to regenerate ADP-p53, thus recycling the high-affinity DNA binding form of p53. The ATP/ADP regulatory mechanism applies to two distinct types of p53 interaction with DNA, namely, sequence-specific DNA binding (via the core domain of the p53 protein) and binding to sites of DNA damage (via the C-terminal domain). Further studies indicate that ADP not only stabilizes p53-DNA complexes but also renders the complexes susceptible to dissociation by specific p53 binding proteins. We propose a model in which the DNA binding functions of p53 are regulated by an ATP/ADP molecular switch, and we suggest that this mechanism may function during the cellular response to DNA damage.     

The effects of cardiolipin on the structural dynamics of the mitochondrial ADP/ATP carrier in its cytosol-open state

Cardiolipin (CL) has been shown to play a crucial role in regulating the function of proteins in the inner mitochondrial membrane. As the most abundant protein of the inner mitochondrial membrane, the ADP/ATP carrier (AAC) has long been the model of choice to study CL-protein interactions, and specifically bound CLs have been identified in a variety of crystal structures of AAC. However, how CL binding affects the structural dynamics of AAC in atomic detail remains largely elusive. Here we compared all-atom molecular dynamics simulations on bovine AAC1 in lipid bilayers with and without CLs. Our results show that on the current microsecond simulation time scale: 1) CL binding does not significantly affect overall stability of the carrier or structural symmetry at the matrix-gate level; 2) pocket volumes of the carrier and interactions involved in the matrix-gate network become more heterogeneous in parallel simulations with membranes containing CLs; 3) CL binding consistently strengthens backbone hydrogen bonds within helix H2 near the matrix side; and 4) CLs play a consistent stabilizing role on the domain 1-2 interface through binding with the R30:R71:R151 stacking structure and fixing the M2 loop in a defined conformation. CL is necessary for the formation of this stacking structure, and this structure in turn forms a very stable CL binding site. Such a delicate equilibrium suggests the strictly conserved R30:R71:R151stacking structure of AACs could function as a switch under regulation of CLs. Taken together, these results shed new light on the CL-mediated modulation of AAC function.     

Conserved structural domains among species and tissues-specific differences in the mitochondrial phosphate-transport protein and the ADP/ATP carrier

Peptide maps were generated of the CNBr-digested mitochondrial phosphate-transport protein and ADP/ATP carrier from bovine and rat heart, rat liver and blowfly flight muscle. Total mitochondrial proteins from the same sources plus pig heart were separated by SDS-polyacrylamide gel electrophoresis. The peptide maps and the total mitochondrial proteins were electroblotted onto nitrocellulose membranes and reacted with rabbit antisera raised against the purified bovine heart phosphate-transport protein and the ADP/ATP carrier. On the basis of antibody specificity, mobility in SDS-polyacrylamide gel electrophoresis, and peptide maps the following was concluded. Phosphate-transport protein alpha and phosphate-transport protein beta (pig and bovine heart) react equally with the first and also with the second of two independent phosphate-transport protein-antisera. Tissue-specific structural domains exist for both the phosphate-transport protein and the ADP/ATP carrier, i.e., one phosphate-transport protein-antiserum reacts with the phosphate-transport protein from all assayed sources, the other only with the cardiac phosphate-transport protein. These differences may reflect tissue-specific regulation of phosphate and adenine nucleotide transport. Homologies among the different species are found for the phosphate transport protein and the ADP/ATP carrier, except for the flight muscle ADP/ATP carrier. These conserved structural domains of the phosphate-transport protein may relate directly to catalytic activity. Alkylation of the purified phosphate-transport proteins and the ADP/ATP carriers by the transport inhibitor N-ethylmaleimide affects electrophoretic mobilities but not the antibody binding. Neither of the two phosphate-transport protein-antisera nor the ADP/ATP-carrier antiserum react with both phosphate transport protein and ADP/ATP carrier, even though these two proteins possess similarities in primary structure and function. Possible mechanisms for generating tissue-specific structural differences in the proteins are discussed.     

Conserved structural domains among species and tissues-specific differences in the mitochondrial phosphate-transport protein and the ADP/ATP carrier

Peptide maps were generated of the CNBr-digested mitochondrial phosphate-transport protein and ADP/ATP carrier from bovine and rat heart, rat liver and blowfly flight muscle. Total mitochondrial proteins from the same sources plus pig heart were separated by SDS-polyacrylamide gel electrophoresis. The peptide maps and the total mitochondrial proteins were electroblotted onto nitrocellulose membranes and reacted with rabbit antisera raised against the purified bovine heart phosphate-transport protein and the ADP/ATP carrier. On the basis of antibody specificity, mobility in SDS-polyacrylamide gel electrophoresis, and peptide maps the following was concluded. (1) Phosphate-transport protein and phosphate-transport protein β (pig and bovine heart) react equally with the first and also with the second of two independent phosphate-transport protein-antisera. (2) Tissue-specific structural domains exist for both the phosphate-transport protein and the ADP/ATP carrier, i.e., one phosphate-transport protein-antiserum reacts with the phosphate-transport protein from all assayed sources, the other only with the cardiac phosphate-transport protein. These differences may reflect tissue-specific regulation of phosphate and adenine nucleotide transport. (3) Homologies among the different species are found for the phosphate transport protein and the ADP/ATP carrier, except for the flight muscle ADP/ATP carrier. These conserved structural domains of the phosphate-transport protein may relate directly to catalytic activity. (4) Alkylation of the purified phosphate-transport proteins and the ADP/ATP carriers by the transport inhibitor N-ethylmaleimide affects electrophoretic mobilities but not the antibody binding. (5) Neither of the two phosphate-transport protein-antisera nor the ADP/ATP-carrier antiserum react with both phosphate transport protein and ADP/ATP carrier, even though these two proteins possess similarities in primary structure and function. Possible mechanisms for generating tissue-specific structural differences in the proteins are discussed.     

Interaction between domains of a plant NBS-LRR protein in disease resistance-related cell death

Many plant disease resistance (R) genes encode proteins predicted to have an N-terminal coiled-coil (CC) domain, a central nucleotide-binding site (NBS) domain and a C-terminal leucine-rich repeat (LRR) domain. These CC-NBS-LRR proteins recognize specific pathogen-derived products and initiate a resistance response that often includes a type of cell death known as the hypersensitive response (HR). Co-expression of the potato CC-NBS-LRR protein Rx and its elicitor, the PVX coat protein (CP), results in a rapid HR. Surprisingly, co-expression of the LRR and CC-NBS as separate domains also resulted in a CP-dependent HR. Likewise, the CC domain complemented a version of Rx lacking this domain (NBS- LRR). Correspondingly, the LRR domain interacted physically in planta with the CC-NBS, as did CC with NBS-LRR. Both interactions were disrupted in the presence of CP. However, the interaction between CC and NBS-LRR was dependent on a wild-type P-loop motif, whereas the interaction between CC-NBS and LRR was not. We propose that activation of Rx entails sequential disruption of at least two intramolecular interactions.     

Molecular genetics of disease resistance in cereals

AIMS: This Botanical Briefing attempts to summarize what is currently known about the molecular bases of disease resistance in cereal species and suggests future research directions. SCOPE: An increasing number of resistance (R) genes have been isolated from rice, maize, wheat and barley that encode both structurally related and unique proteins. This R protein diversity may be attributable to the different modus operandi employed by pathogen species in some cases, but it is also a consequence of multiple defence strategies being employed against phytopathogens. Mutational analysis of barley has identified additional genes required for activation of an R gene-mediated defence response upon pathogen infection. In some instances very closely related barley R proteins require different proteins for defence activation, demonstrating that, within a single plant species, multiple resistance signalling pathways and different resistance strategies have evolved to confer protection against a single pathogen species. Despite the apparent diversity of cereal resistance mechanisms, some of the additional molecules required for R protein function are conserved amongst cereal and dicotyledonous species and even other eukaryotic species. Thus the derivation of functional homologues and interacting partner proteins from other species is contributing to the understanding of resistance signalling in cereals. The potential and limit of utilizing the rice genome sequence for further R gene isolation from cereal species is also considered, as are the new biotechnological possibilities for disease control arising from R gene isolation. CONCLUSIONS: Molecular analyses in cereals have further highlighted the complexity of plant-pathogen co-evolution and have shown that numerous active and passive defence strategies are employed by plants against phytopathogens. Many advances in understanding the molecular basis of disease resistance in cereals have focused on monogenic resistance traits. Future research targets are likely to include less experimentally tractable, durable polygenic resistances and nonhost resistance mechanisms.     

Salicylic acid: an old hormone up to new tricks

Salicylic acid (SA) acts as a signalling molecule in plant defence against biotrophic and hemibiotrophic phytopathogens. The biosynthesis of SA on pathogen detection is essential for local and systemic acquired resistance, as well as the accumulation of pathogenesis‐related (PR) proteins. SA biosynthesis can occur via several different substrates, but is predominantly accomplished by isochorismate synthase (ICS1) following pathogen recognition. The roles of BTB domain‐containing proteins, NPR1, NPR3 and NPR4, in SA binding and signal transduction have been re‐examined recently and are elaborated upon in this review. The pathogen‐mediated manipulation of SA‐dependent defences, as well as the crosstalk between the SA signalling pathway, other plant hormones and defence signals, is also discussed in consideration of recent research. Furthermore, the recent links established between SA, pathogen‐triggered endoplasmic reticulum stress and the unfolded protein response are highlighted.     

Cooperative binding of ATP and RNA substrates to the DEAD/H protein DbpA

Unlike most DEAD/H proteins, the purified Escherichia coli protein DbpA demonstrates high specificity for its 23S rRNA substrate in vitro. Here we describe several assays designed to characterize the interaction of DbpA with its RNA and ATP substrates. Electrophoretic mobility shift assays reveal a sub-nanomolar binding affinity for a 153 nucleotide RNA substrate (R153) derived from the 23S rRNA. High affinity RNA binding requires both hairpin 92 and helix 90, as substrates lacking these structures bind DbpA with lower affinity. AMPPNP inhibition assays and ATP/ADP binding assays provide binding constants for ATP and ADP to DbpA with and without RNA substrates. These data have been used to describe a minimal thermodynamic scheme for the binding of the RNA and ATP substrates to DbpA, which reveals cooperative binding between larger RNAs and ATP with cooperative energies of approximately 1.3 kcal mol(-1). This cooperativity is lost upon removal of helix 89 from R153, suggesting this helix is either the preferred target for DbpA's helicase activity or is a necessary structural element for organization of the target site within R153.     

The grateful dead: calcium and cell death in plant innate immunity

Plant cells sensing pathogenic microorganisms evoke defence systems that can confer resistance to infection. This innate immune reaction can include triggering of basal defence responses as well as programmed cell death, or hypersensitive response (HR). In both cases (basal defence and HR), pathogen perception is translated into elevated cytosolic Ca(2+) (mediated by plasma membrane and intracellular channels) as an early step in a signalling cascade. Cyclic nucleotide-gated channels contribute to this influx of Ca(2+) into the cell. The molecular nature of other transport proteins contributing to the Ca(2+) elevation is unclear. Pathogen recognition occurs at two levels: the perception of pathogen-associated molecular pattern (PAMP) molecules widely present in microorganisms, and an interaction between pathogen avirulence gene products (if present) and corresponding plant R (resistance) gene products. The Ca(2+) elevation occurring in response to PAMP perception or R gene interactions could occur due to phosphorylation events, G-protein signalling and/or an increase in cyclic nucleotides. Downstream from the initial Ca(2+) rise, the signalling cascade includes: activation of calmodulin and protein kinases, and nitric oxide and reactive oxygen species generation. Some of these downstream events amplify the Ca(2+) signal by further activation of Ca(2+) transporters.     

The Tomato Nucleotide-binding Leucine-rich Repeat Immune Receptor I-2 Couples DNA-binding to Nucleotide-binding Domain Nucleotide Exchange

Plant nucleotide-binding leucine-rich repeat (NLR) proteins enable plants to recognize and respond to pathogen attack. Previously, we demonstrated that the Rx1 NLR of potato is able to bind and bend DNA in vitro. DNA binding in situ requires its genuine activation following pathogen perception. However, it is unknown whether other NLR proteins are also able to bind DNA. Nor is it known how DNA binding relates to the ATPase activity intrinsic to NLR switch function required to immune activation. Here we investigate these issues using a recombinant protein corresponding to the N-terminal coiled-coil and nucleotide-binding domain regions of the I-2 NLR of tomato. Wild type I-2 protein bound nucleic acids with a preference of ssDNA ≈ dsDNA > ssRNA, which is distinct from Rx1. I-2 induced bending and melting of DNA. Notably, ATP enhanced DNA binding relative to ADP in the wild type protein, the null P-loop mutant K207R, and the autoactive mutant S233F. DNA binding was found to activate the intrinsic ATPase activity of I-2. Because DNA binding by I-2 was decreased in the presence of ADP when compared with ATP, a cyclic mechanism emerges; activated ATP-associated I-2 binds to DNA, which enhances ATP hydrolysis, releasing ADP-bound I-2 from the DNA. Thus DNA binding is a general property of at least a subset of NLR proteins, and NLR activation is directly linked to its activity at DNA.     

Structural insight into mutations at 155 position of valosin containing protein (VCP) linked to inclusion body myopathy with Paget disease of bone and frontotemporal Dementia

Mutations in Valosin-containing protein (VCP) have been implicated in the pathology linked to inclusion body myopathy, paget disease of bone and frontotemporal dementia (IBMPFD). VCP is an essential component of AAA-ATPase superfamily involved in various cellular functions. Advanced In-silico analysis was performed using prediction based servers to determine the most deleterious mutation. Molecular dynamics simulation was used to study the protein dynamics at atomic level. Molecular docking was used to study the effect of mutation on ATP/ADP transition in the kinase domain. This ATPase of 806 amino acids has four domains: N-terminal domain, C-terminal domain and two ATPase domains D1 and D2 and each of these domains have a distinct role in its functioning. The mutations in VCP protein are distributed among regions known as hotspots, one such hotspot is codon 155. Three missense mutations reported in this hotspot are R155C, R155H and R155P. Potentiality of the deleteriousness calculated using server based prediction models reveal R155C mutation to be the most deleterious. The atomic insight into the effect of mutation by molecular dynamics simulation revealed major conformational changes in R155C variants ATP binding site in D1 domain. The nucleotide-binding mode at the catalytic pocket of VCP and its three variants at codon 155 showed change in the structure, which affects the ATP–ADP transition kinetics in all the three variants.     

A kinesin switch I arginine to lysine mutation rescues microtubule function

Switch I and II are key active site structural elements of kinesins, myosins, and G-proteins. Our analysis of a switch I mutant (R210A) in Drosophila melanogaster kinesin showed a reduction in microtubule affinity, a loss in cooperativity between the motor domains, and an ATP hydrolysis defect leading to aberrant detachment from the microtubule. To investigate the conserved arginine in switch I further, a lysine substitution mutant was generated. The R210K dimeric motor has lost the ability to hydrolyze ATP; however, it has rescued microtubule function. Our results show that R210K has restored microtubule association kinetics, microtubule affinity, ADP release kinetics, and motor domain cooperativity. Moreover, the active site at head 1 is able to distinguish ATP, ADP, and AMP-PNP to signal head 2 to bind the microtubule and release mantADP with kinetics comparable with wild-type. Therefore, the structural pathway of communication from head 1 to head 2 is restored, and head 2 can respond to this signal by binding the microtubule and releasing mantADP. Structural modeling revealed that lysine could retain some of the hydrogen bonds made by arginine but not all, suggesting a structural hypothesis for the ability of lysine to rescue microtubule function in the Arg210 mutant.